This invention relates to assay methods for detecting undesired microorganisms, e.g., Salmonella, in samples, e.g., of food products.
It has long been known that it is beneficial to test for microorganisms to assess the safety and quality of raw materials and finished products, the hygiene within production environments, the efficacy of physical and chemical preservation processes, the microbiological stability of finished products (shelf-life testing), and to meet safety and quality specifications.
However, conventional tests for microorganisms are typically labor intensive and material expensive, often have low precision and accuracy, and need long incubation periods. Results for conventional test are often obtained only after the raw materials have been used, perishable products have been distributed, or short shelf-life products have been consumed. Consequently, it is important to perform such tests as rapidly, accurately, precisely, and economically as possible.
One major concern relating to food products is Salmonella contamination. The genus Salmonella includes many serovars that cause enteric disease in man. The ingestion of salmonella-contaminated food can cause food poisoning that may be fatal. Standard tests for Salmonella can take more than a week to demonstrate that a sample is free from contamination. Another problem with conventional tests is that Salmonella serotypes are a large physiologically diverse group that require a range of media to be used for their detection. Typical conventional tests to detect Salmonella in food include resuscitation or preenrichment in liquid media, enrichment, i.e., selective culture, in liquid media, isolation of individual colonies on selective and differential agars, and confirmation of identity using biochemistry and serology techniques.
Recently, nucleic acid probes have been utilized to test for microorganism contamination. Such probes are designed to hybridize preferentially with the DNA or RNA of specific target organisms that may be present in a sample and are typically labelled so that they can be detected if they hybridize with the target microorganism. Samples are inoculated into liquid growth media in which the target microorganisms, if any, can propagate. Thereafter, the nucleic acid probes are added under conditions that allow preferential hybridization, and the hybridization products, if any, are detected by various assays, e.g., colorimetric assays.
Such nucleic acid probe test methods are accurate and quick, but the assays for the hybridization products can be hampered by the growth media in which the target microorganisms are propagated.
The European marketplace typically utilizes microorganism enrichment strategies different than those used in the U.S. For example, the U.S. market typically enriches according to methodology described in the Bacterial Analytical Manual (BAM), an FDA publication, e.g., using selenite cysteine and tetrathionate-Brilliant Green broths for the enrichment of Salmonella from foods. The Europeans, however, use RV medium almost exclusively for this purpose. RV medium is composed of the following ingredients per liter of water: 5.0 g soya peptone, 8.0 g NaCl, 1.6 g KH.sub.2 PO.sub.4, 40.0 g MgCl.sub.2, and 0.4 g Malachite Green (pH=5.2.+-.0.2).
However, this RV medium has been shown to be detrimental to the use of DNA probe hybridization reactions. For example, if a food sample containing Salmonella is enriched according to BAM methodology, the endpoint signal, in a colorimetric assay for hybridization products is expected to be between 1.0 and 2.0 O.D. units (at 450 nm). However, when the same sample is enriched using RV medium, the endpoint signal for the same assay is between 0.0 and 0.1 O.D. units, with many samples being read as false negatives.
The inventors have developed a solution to this low signal problem for such assays, in particular, to perform in the European marketplace.